1. Field of the Invention
The invention relates to a monoclonal antibody (mAb) against human complement receptor type 2 (CR2, CD21) and a hybridoma as well as process to obtain the same. Furthermore, the invention is directed to therapeutic applications thereof.
2. Description of the Related Arts
Human CR2 (CD21) is a membrane glycoprotein of 145 kDa predominantly expressed on mature B lymphocytes (Tedder, T. F., L. T. Clement, and M. D. Cooper. 1984. Expression of C3 d receptors during human B cell differentiation: immunofluorescence analysis with the HB5 monoclonal antibody. J. Immunol. 133:678) and follicular dendritic cells (Reynes, M., J. P. Aubert, J. H. Cohen, J. Audouin, V. Tricottet, J. Diebold, and M. D. Kazatchkine. 1985. Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens. J. Immunol. 135:2687), and to a much lesser extent on peripheral blood T lymphocytes, thymocytes or astrocytes. The function of CR2 has been most extensively studied for B-lymphocytes (for a review, see Fearon, D. T. and R. H. Carter. 1995. The CD19/CR2/TAPA-1 complex of B lymphocytes: Linking natural to acquired immunity. Annu. Rev. Immunol. 13:127). CR2 is the receptor for special CR2-binding fragments, in particular for C3dg and (with lower affinity) for iC3b, fragments of C3 that are generated during complement activation and covalently deposited on surfaces e.g. of pathogens. On B-cells, CR2 forms a non-covalent receptor complex with the B-cell specific CD19 molecule and CD81 which is broadly expressed among hematopoietic cells. Coligation of this complex and the BCR-complex lowers the threshold for B-cell activation substantially. The extent of this effect is exponentially correlated to the number of C3dg-residues on the specific antigen.
Of pathophysiological relevance, Epstein-Barr virus infection of human cells is known to require attachment to CR2 as a first step.
CR2 is a member of the family of C3b-binding and/or C4b-binding proteins and its extracellular part is formed by 15 or, as a result of alternative splicing, 16 short consensus repeats (SCR). These structural units of about 60 amino acids share a frame of several strictly conserved amino acids, most importantly four cysteine residues which are linked by disulfide bonds in a way that the first and third Cys and the second and fourth Cys form a bond, respectively.
Characterization of the two N-terminal SCRs involved in ligand binding has extensively made use of the mouse mAb OKB7 which was shown early to inhibit CR2-dependent EAC3d-rosetting of Raji cells (Rao, P. E., S. D. Wright, E. F. Westberg, and G. Goldstein. 1985. OKB7, a monoclonal antibody that reacts at or near the C3d binding site of human CR2. Cell. Immunol. 93:549) and to reduce infection of B-cells with EBV (Nemerow, G. R., R. Wolfert, M. E. McNaughton, and N. R. Cooper. 1985. Identification and characterization of the Epstein-Barr virus receptor on human B lymphocytes and its relationship to the C3d complement receptor (CR2). J. Virol. 55:347). OKB7 has also been employed to demonstrate an epitope dependence regarding effects of CR2-mAbs on B-cell proliferation. However, this feature of OKB7 versus other non-blocking mAbs as HB5 (Tedder, T. F., L. T. Clement, and M. D. Cooper. 1984. Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB5 monoclonal antibody. J. Immunol. 133:678) is not consistently defined.
Although mAb OKB7 inhibited EAC3d-rosetting with CR2-expressing cells to &gt;95% in our hands, it was rather inefficient in blocking CR2-dependent complement activation on Raji cells. It is, therefore, an object of the invention to generate more effective mAbs. Further objects are the generation of corresponding hybridomas and therapeutic applications. To this end, employing a baculovirus-derived soluble CR2-protein truncated after SCR 4 (Prodinger, W. M., J. Schoch, M. G. Schwendinger, J. Hellwage, W. Parson, P. F. Zipfel, and M. P. Dierich. 1997. Expression in insect cells of the functional domain of CD21 (complement receptor type two) as a truncated soluble molecule using a baculovirus vector. Immunopharmacology 38:141) as an immunogen and inhibition and removal of the binding of FITC-labelled, C3d-coated agarose microbeads to CR2 as a screening method proved to be a successful strategy.